Recombinant varicella-zoster virus and process for constructing same

ABSTRACT

The present invention provides a recombinant varicella-zoster virus prepared by inserting, into the viral genome, nucleic acids from the hepatitis B virus genome, a genomic DNA of the recombinant varicella-zoster virus, a live vaccine containing the recombinant varicella-zoster virus as an effective ingredient, an antigen derived from the recombinant varicella-zoster virus, and diagnostic agent containing the antigen. 
     The recombinant varicella-zoster virus of the present invention can be utilized as a multivalent vaccine having an excellent immunity effect both on chicken pox and hepatitis B, and expression products and genomic DNA thereof may be used as a multivalent diagnostic agent.

This is a divisional application of Ser. No. 08/340,880 filed Nov. 15,1994, now U.S. Pat. No. 5,653,976, which is a continuation of nowabandoned application Ser. No. 07/873,821 filed Apr. 27, 1992, nowabandoned.

FIELD OF THE INVENTION!

The present invention relates to a recombinant varicella-zoster virusand a process for constructing same, the recombinant varicella-zostervirus thus obtained and the antigens and genomic DNA thereof beingapplicable as a vaccine, an immunological diagnostic agent, a geneticdiagnostic agent, and a genetic engineering reagent.

PRIOR ART!

The technology for constructing a recombinant virus by inserting foreigngenes or heterogenes into viral genes, i.e., the technology to use theviral genome as cloning and/or expression vector has been employed since1979, for example, in the production of rabbit β-globin using SV40 as aviral vector Nature (London), 277, 108-114, 1979, ibid., 278, 35-40,1979!. In 1980,the general meeting of the World Health Organization(WHO) declared extermination of smallpox based on the successful resultsof vaccine and recommended abolition of vaccination. Since then, theeffective use of vaccinia virus which is an attenuated virus forming aneffective ingredient of vaccination has attracted general attentionthroughout the world and is revaluated at present. Under suchcircumstances, a recombinant vaccinia virus created for the purpose ofutilizing said viral genome as the cloning and expressions vector offoreign genes has been reported Proceedings of National Academy ofScience (U.S.A.), 79, 4927-4931, 1982; ibid., 79, 7415-7419, 1982!. Inaddition, the ad hoc consulting group for WHO adopted aresearch-promoting plan for a recombinant virus vaccine using vacciniavirus, etc as a vector. Nature (London), 312, 299, 1984!. This WHOproposal suggests: developing a viral vector such as a vaccinia virusvector with a wider host range which can utilize a higher animal cell asa host, with a view to solving the drawback of the conventionally usedplasmid vector, phage vector and cosmid vector with a narrow hostrange(mainly bacteria or yeasts); and developing a multivalent vaccineusing, as an effective ingredient, a recombinant virus constructed byinserting at least two kinds of foreign genes into vaccinia virus genomein place of the conventional combined vaccine comprising two or morekinds of antigens or heteroviruses. With the abovementioned WHOdeclaration and proposal as the starting point, fundamental research anddevelopment efforts regarding viral vectors have actively been made invarious fields, and as a result, a huge volume of data has already beenaccumulated. More particularly, viral genomes already known to serve assuch viral vectors include, for example, papillomavirus, polyomavirus,adenovirus, retrovirus, vacuolovirus, herpes simplex virus, Marek'sdisease virus, varicella virus, parbovirus, cauliflower mosaic virus,tobacco mosaic virus, and tomato golden mosaic virus Virus, 36, 1-41,1986; ibid., 37, 1-40 1987; "Current Communication in Molecular Biology:Viral Vector," pp. 10198, Y. Olusman and S. H. Hughes (ed.), Cold SpringHarbor Laboratory (U.S.A.) pub. 1988, European Patent ProvisionalPublication No. 334,530!.

From among the above-listed viral vectors, the varicella-zoster virus(hereinafter abbreviated as "VZV" ) related to the present invention isdescribed below.

VZV is usually a cell-associated virus, the infectivity of which can bemaintained only within live cells, so that it is difficult to separatethe virus and subculture and mass-produce it in a test tube. There hastherefore been a general tendency of to be hesitant not only aboutmaking efforts to develop a VZV vaccine and diagnostic agents but alsoabout fundamental and clinical research efforts on VZV as a pathogen ofchicken pox. Progress of studies on VZV has therefore been very slowuntil the establishment of the cell-free Oka strain of attenuated VZVfor live varicella vaccine by the present inventors in 1975 BikenJournal, 23, 53-55, 1975; Japanese Patent Publication No. 51-19,018,Japanese Patent Publication No. 53-41,292; Japanese Patent PublicationNo. 56-42,144). Taking advantage of the development of a live vaccineusing the above-mentioned Oka strain of attenuated VZV, fundamental andclinical research and applied research efforts on VZV have actively beencarried out at various localities in the world. The live varicellavaccine using this Oka strain of attenuated VZV as the effectiveingredient is thus now widely applied in practice throughout the worldRequirements for Varicella Vaccine (Live) Adopted 1984; WHO TechnicalReport Series, No. 725, pp. 102-124, 1985!. A huge volume of dataregarding fundamentals, clinical, diagnosis and immunology of VZV hasalready been accumulated "Virology," vol. 2, pp. 2011-2054, B. N.Fields, et al. (ed.), Raven Press (U.S.A.), pub. 1990!. Particularly,progress in the structural analysis of VZV genome has made great stridessince the beginning of the 1980s through restriction mapping and totalbase sequence determination of VZV genome DNA, along with thedevelopment and diffusion of the technique for cloning of genes andexpression thereof, and monoclonal antibody (Journal of GeneralVirology, 67, 1759-1816, 1986; ibid., 67, 1817-1829, 1986; Virus, 37,71-80, 1987). Some major virological findings as to VZV as derived fromthe references as referred to above are as follows.

VZV has an envelope, is a DNA-type virus taxologically falling withinthe herpes simplex virus group, i.e., herpesviridae,alphaherphesvirinae, and has a dia. of approximately 180 to 200 nm. Thisgenome consists of a linear DNA having two chains of approximately 120kilobase(kb), and is contained in a doughnut-shaped core having a dia.of approximately 75 nm within the nucleocapsid. This genemc DNAcomprises an unique sequence (U) consisting of a long segment (U_(L))and a short segment (U_(S)), a terminal repeat sequence (TR) consistingof TR_(L) and TR_(S) adjacent to U_(L) and U_(S), respectively, and aninverted repeat sequence (IR) consisting of IR_(L) and IR_(S)complementary to TR_(L) and TR_(S), respectively. These six segments arearranged in the direction from 5' toward 3' end in the order of TR_(L),U_(L), IR_(L),IR_(S), U_(S) and TR_(S). Currently, 71 ORFs (open readingframes) In total are numbered from 1 to 71 starting from the 5' end, andfrom among these genes, functions of the following 21 ORFs areidentified or estimated the figures in parentheses representing the ORFNos.!: (4) early protein, (8) deoxyurinetriphosphatase, (10)trans-inducible protein, (13) thymidine synthetic enzyme, (14)glycoprotein (hereinafter abbreviated as "gp") V, (18) polynucleotidereductase small subunit, (19) polynucleotide reductase large subunit,(28) DNA ligase, (29) DNA-binding protein, (31) gp II, (36) thymidinekinase (hereinafter abbreviated as "tk"), (37) gpIII, (40) capsidprotein, (48) exonulease, (62, 71) early protein, (63, 70) earlyprotein, (66) protein kinase, (67) gpIV, and (68) gpI.

The following recombinant VZVs constructed by using VZV as a viralvector are known: a recombinant VZV obtained by inserting a foreign geneinto the downstream region of the gpI promoter gene of VZV, the foreigngene being gp 350 of Epstein-Barr virus (Japanese Patent ProvisionalPublication No. 12,277/88 or European Patent Provisional Publication No.251,534; Japanese Patent Provisional Publication No. 141,589/88; Journalof Virology, 61, 1796-1867, 1987); one having preS2 of hepatitis B virus(hereinafter abbreviated as "HBV") and surface antigen (hereinafterabbreviated as "HBs") gene (Japanese Patent Provisional Publication No.12,277/88 or European Patent Provisional Publication No. 251,534) asforeign genes; a recombinant VZV prepared by combining gp300 and gp220genes of Epstein-Barr virus into the downstream of the VZV gpI promotergene and inserting further the resultant gene fragments into VZV tk geneso that the Epstein-Barr virus genes may be expressed as fusion proteinwith tk Proceedings of National Academy of Science U.S.A.!, 84,3896-3900, 1987; "ULCA Symposia on Molecular Biology; New Series, vol.84: Technical Advances of Vaccine Development," pp. 235-241, by R. W.Ellis, at al., Alan R. Liss, Inc. (U.S.A.) pub. 1988!. However, allthese recombinant VZVs are low in immunogenicity, and the safety andeffectiveness thereof have not as yet been confirmed. No attempt hasbeen reported to the present of putting on the market any vaccines ordiagnostic agents prepared using these VZVs as antigens. The recombinantVZVs have not yet reached the level of practical applicaton.

The conceivable reasons are as follows: (1) as compared with a virus ofsmall size having usually a genome length of approximately 10 kb, a VZVis large with a very long infectious genome length of approximately 120kb, and such a long DNA chain is susceptible to breakage and istherefore unstable; (2) a VZV, which is originally a cell-dependentvirus, very unstable to heat, requires storage at a temperature lowerthan -60° C. for retaining the level of infectivity, and handling,culturing and mass production thereof are not easy; and (3) the numberof produced infectious viral particles per cell in viral culture, whichis approximately 10 to 100 particles for the other viruses, is as low asapproximately 0.1 particle for VZV, leading to a very low productionyield. For these reasons, it is very difficult to prepare or clone agenomic DNA of VZV which is suitably applicable not only for massproduction of the genomes through VZV culturing but also in the test andresearch stage, and there is only a very low probability of screening arecombinant VZV eligible for industrial use in quality as well as inquantity. That is, the establishment of a recombinant VZV requiresovercoming these very difficult conditions.

SUMMARY OF THE INVENTION!

The objective of the present invention is to provide a novel recombinantVZV having a high expression ability of foreign genes inserted thereinand a genetic stability of such ability through succeeding generations,and being safe and effective as an ingredient of a live vaccine. Moreparticularly, the objective of the present invention is to provide arecombinant VZV carrying HBV genes which were particularly difficult toculture and mass-produce with other host-vector systems, from the pointof view of practical use.

The present invention provides a recombinant VZV constructed byinserting at least one kind of gene selected from a gene group of HBVgenome into a downstream region of a promoter gene of VZV and a processfor constructing same.

The present invention also provides a genomic DNA of the above-mentionedrecombinant VZV, a live vaccine containing this recombinant VZV, anantigen derived from this recombinant VZV, and diagnostic agentscontaining this antigen.

BRIEF DESCRIPTION OF THE DRAWINGS!

FIG. 1 is a schematic representation of the construction of arecombinant VZV of the present invention. The process A iscotransfection of VZV DNA and chmeric plasmid DNA(pHH), and B isrecombination screening by IFA test;

FIG. 2 is a schematic representation of DNA base sequences of the HBVpreS2-HBs gene and the VZV tk gene, and of the recombinant sequence ofthese genes. Indications: Sta*, starting codon of pre S2 gene; Sta**,starting codon of HBs gene; Sta***, starting codon of VZV tk gene; Ter,termination codon of HBs gene; X*, base for VZV tk gene; X', base forflesh end with T4 DNA polymerase; and X⁺, base for HBs gene; and

FIG. 3 shows purification of HBs secreted from cells infected with therecombinant VZV of the present invention. HBs antigen secreted frominfected cells was concentrated and subjected to equilibriumcentrifugation in CsCl. The density ( ) of the fraction was monitored bymeasuring the volume and weight, and titers of HBs antigen ( ) wereassayed by the RPHA test.

DETAILED DESCRIPTION OF THE INVENTION!

The configuration and preferred embodiments of the present invention areas follows.

VZV strain as a viral vector

In the present invention, which has as a main object to create arecombinant live vaccine excellent in safety, effectiveness anduniformity, the VZV genome is used as a vector. According to the presentinvention, any of such existing known VZV strains as those of Oka (ATCCVR-795), Webster (ATCC VR-916), Ellen (ATCC VR-586), YS, YG, SCOll,Kawaguchi and Dumas (Journal of General Virology, 67, 1816-1829, 1986;Virus, 37, 71-80, 1987) and VZV strains to be isolated may be used. Fromamong these VZV strains, it is preferable to use an attenuated VZVstrain with a view to ensuring a high safety in handling during test andresearch and during manufacture, and achieving industrial use includingsupply of a live vaccine and diagnostic agents excellent in both safetyand effectiveness. Particularly from the point of view of the mainobject of the present invention presented above, the Oka strain ofattenuated VZV, which is the only DNA-type attenuated virus establishedand publicly recognized worldwide for excellent safety andeffectiveness, and currently employed throughout the world, is mostsuitable for the present invention.

Culture of VZV and selection of cells

Any of the conventional cells having a known sensitivity to VZV derivedfrom human, monkey and guinea pig may be used. From among these cells,on account of the probability of mixture of aberratic and caricinogenicfactors, the eligibility as cells for culture of viral strain forvaccine, and the quantity of produced viruses, it is desirable to usehuman diploid cells such as HEL299 (ATCC No. CCL 137), MRC-5 (ATCC No.CCL 171), and WI-38 (ATCC No. CCL 75) publicly recognized for themanufacture of live vaccines. As the culture medium for mutiplicationand maintenance of these cells and for culturing viruses, a commerciallyavailable synthetic culture medium such as 199 Culture Mediummanufactured by Difco Co. (U.S.A.)!, or MEM Culture Medium manufacturedby Gibco Co. (U.S.A.)! may be used. Such a culture medium is applied forculturing by adjusting pH to approximately 6.8 to 8.0 through dropaddition and mixture of approximately 7% (w/v) aqueous solution ofNaHCO₃, and then adding fetal bovine serum for example, thatmanufactured by Flow Co. (U.S.A.)! so as to achieve a finalconcentration of approximately 2 to 15% (w/v) immediately before use.VZV is cultured on a maintenance culture medium after inoculating a seedvirus to previously prepared cell culture. The recommended temperaturefor culturing is within the range between 25° to 40° C., or morepreferably, within the range of from 33°to 38° C.

Selection of VZV gene into which HBV genes are to be inserted

Theoretically, according to the present invention, all theabove-mentioned 71 VZV genes can be used as portions into which HBVgenes are to be inserted, respectively. Precautions to be taken in thisuse are to insert an HBV gene into the ORF region located in thedownstream of the promoter gene thereof so as to permit functioning ofthe promoter gene, and to accomplish insertion without destroying themutual codons of the VZV gene and the foreign gene so as to ensuresmooth translation. In general, since a viral gene into which a foreigngene has been inserted may be destructed and become unable to performits original functions, it is desirable to adopt, as the promoter genefor combination of HBV gene, a gene which does not exert an influence oninfectivity and multiplication of virus and has high expression abilitywith a strong promoter intensity, such as tk gene or gp gene.Particularly when using Oka strain of attenuated VZV as a viral vector,as the lack or the degradation of expression ability of the gpV gene(14th ORF) is known (Journal of Virology, 64, 4540-4548, 1990), use ofthis gene region cannot be recommended for an insertion site. It ishowever possible to use the gpV gene of a wild-type VZV; by inserting anHBV gene into the downstream of the promoter gene thereof, it ispossible to cause destruction of the gene region encoding said gpV or todecrease the amount of expression. The recombinant VZV obtainedtherefrom is expected to show a very high usefulness as an attenuatedstrain similar to that of the Oka grain of attenuated VZV.

Selection of HBV genes to be inserted into VZV genome

Theoretically, according to the present invention, it is possible touse, as foreign genes to be inserted, one or more kinds of gene selectedfrom the gene group of HBV genome to the extent or not deterioratingmultiplication and infectivity of VZV. It is however necessary to becareful to prevent the gene regions from encoding toxic substances, orfrom detrimental substances and toxins involved in foreign genes, withdue regard to the use of the recombinant VZV as a live vaccine. Forexample, both the preS1 region of HBV and a part of the preS2 regionwhich is a polymerized human serum albumin receptor are known to have ahigh tropism for hepatic cells. When using a portion near the geneencoding any of these regions as a foreign gene, therefore, it isnecessary to eliminate said region by means of a restriction enzyme. Therecombinant VZV into which HBV genes have thus been inserted possessesantigenicity and immunogenicity for both HBV and VZV, as it is anattenuated strain, it is applicable in practice as an effectiveingredient of a divalent live vaccine capable of providing immunitysimultaneously against hepatitis B and chikenpox, and as a divalentantigen for diagnosis comprising both HBV and VZV antigens. It shouldparticularly be noted here that, in the present invention, it ispossible to put to practical use, as a live vaccine, an antigenavailable only as an inactivated vaccine in the conventional technology,such as an HBV antigen.

Construction of recombinant VZV

This process may be accomplished in the following sequence of steps:cloning VZV genes into which foreign genes are to be inserted; cloningthe foreign genes; preparing chimeric plasmid formed by inserting theforeign genes into the thus cloned VZV gene; and constructing arecombinant VZV through recombination between this chimeric plasmid andthe VZV genome. These steps are described below in this order.

(1) Cloning of VZV gene: After extracting and purifying VZV genomic DNAfrom cells infected therewith in the manner as described in Example 1presented later, this DNA is digested by means of restriction enzymes,and these DNA fragments prepared through fractionation by theapplication of the conventional agarose gel electrophoresis are clonedwith the use of a commercially available or known host-vector system (P.H. Powels, "Cloning Vector--A Laboratory Manual," 2 vols., Elsevier,1985; "ATCC Recombinant DNA Materials," American Type CultureCollection, 1989). It is possible to clone, for example, VZV tk gene byinserting an H fragment prepared through digestion of a VZV genome withrestriction enzyme SacI (Intervirology, 29, 301-310, 1988) into the Sacdsite of E. coli plasmid vector pUC12 Methods in Enzymology, vol. 101,pp. 20-78, Academic Press (U.S.A.), 1983! and then introducing theplasmid to E. coli JM 109 strain (ATCC No. 53323) for transformation. Analready cloned VZV gene (for example, Journal of General Virology, 67,1817-1829, 1986) may be utilized for simplifying this process.

(2) Cloning of HBV gene: Solution with a detergent or a ultrasonic wavesof extracted cells of HBV-infected tissue or cultured HBV-infectedcells, supernatant of viral culture or blood of an infected subject maybe used as a starting material. A combination of known methods may beused for purifying the material. A purified fraction of virus particlesis first prepared from the starting material by the use of a combinationof, for example, the ammonium sulfate salting-out method, lowspeedcentrifugation, and/or the density gradient ultracentrifugation.Extraction and purification of genomic DNA from this purified fractionshould preferably be carried out at a pH of 3 to 10 to avoidirreversible denaturation thereof, and may be conducted by theconventional method. Genomic DNA may be prepared by adopting anappropriate combination of, for example, the heat/salt method using NaClsolution at 100° C., the detergent method using SDC (sodiumdeoxycholate) or SDS (sodium dodecylsulfate), the phenol extractingmethod based on the principle of diphasic distribution, the guanidinechloride method using a concentrated liquid of guanidine chloride, thealkali method applied at a pH of approximately 10 with the use of NaOHsolution or Na₂ Co₃ --NaHCO₃ buffer solution, and/or the alcoholprecipitation method using cold ethanol. This DNA can be cloned in thesame manner as in (1) above. To simplify the process, an already clonedHBV gene may be employed. For example, the HBV preS1-preS2-HBs antigengene cloned in pM1B11 Japanese Patent Provisional Publication No.22,098/88 (Biken No. 1081)! is applicable.

(3) Preparation of chimeric plasmid of VZV-HBV gene: The downstreamregion of the VZV gene within the plasmid vector prepared in (1) aboveIs digested with restriction enzyme, the DNA fragment derived from theHBV gene cloned in (2) above is then inserted into the restriction siteof said plasmid vector, and this plasmid is transfected to the host cellfor transformation. A chimeric plasmid is thus constructed with linkagebetween the VZV gene and the HBV gene.

(4) Construction of recombinant VZV: A recombinant VZV is constructed byintroducing simultaneously the DNA fragment of VZV genome in (1) aboveand the chimeric plasmid prepared in (3) above into the host cell tocause recombination therewith. For the introduction of the DNA fragmentand the chimeric plasmid DNA into host cells, any of the known methodsincluding the DEAE dextran method, the electroporation method and/or thecalcium phosphate method is applicable. In the case of the calciumphosphate method (Virology, 52, 465-467, 1973), coprecipitate of bothDNAs is prepared coexistently with phosphate ions and calcium ions, andthen the coprecipitate is brought into contact with the cell culture tocause cotransfection. Coprecipitate of calcium phosphate with chimericplasmid DNA is brought into contact by drop addition of infected cellscultured for a period of approximately two to three hours by previouslyinoculating the VZV parental strain, thus introducing said DNA intoVZV-infected cells. Then, a recombinant VZV can be created by culturingthese cells, and causing recombination within this culture systembetween the VZV genomic DNA and the chimeric plasmid DNA.

(5) Recombinant VZV and culture thereof: All the various recombinant VZVclones available in the present invention are attenuated VZV strains andapplicable as an effective ingredient of a live vaccine. For example,recombinant VZV clones between VZV and HBV genes are generically namedas rVH Oka strain series, as disclosed in Example 6 presented later, andconsist of the following 20 named strains: rVH1 Oka strain, and rVH2,rVH3, rVH4, rVH5, rVH6, rVH7, rVH8, rVH9, rVH10, rVH11, rVH12, rVH13,rVH14, rVH15, rVH16, rVH17, rVH18, rVH19 and rVH20 Oka strains. Therecombinant VZV of the present invention may be obtained by inoculatinga recombinant VZV seed virus into a cultured cell in the same manner asin Example 1 presented later and culturing same. In the culturesupernatant and infected cells, there are produced, in addition toinfectious recombinant VZV particles, various antigens which are theexpression products of recombinant VZV genome. For example, by culturingthe above-mentioned rVH7 Oka strain which was deposited under theBudapest Treaty in the European Collection of Animal Cell Cultures(ECACC) whose address is PHLS Centre for Applied Microbiology, PortonDown, Salisbury Wilts, SP4 OJG, U.K., on Apr. 15, 1992 and registered asrVH17-5 `Oka` strain (Provisional Accession No. V92041523), HBspolypeptides having molecular weights of 30 k, 35 k, etc. are producedin the culture supernatant, and HBs polypeptides of 26 k, 30 k, etc., inthe infected cells, in addition to recombinant VZV particles. All theseantigens and viral particles produced in the culture product ofrecombinant VZV and DNA thereof are applicable as vaccines,immunological diagnostic agents, generical diagnostic agents or geneticengineering reagents.

Restriction enzyme analysis of recombinant VZV genomic DNA

Analysis is possible by a conventional method such as the southern blotmethod (Journal of Molecular Biology, 98, 503, 1975). More specifically,a DNA fragment prepared through digestion of a VZV genome by means ofrestriction enzyme is fractioned by agarose gel electrophoresis, andthese fractions are transferred onto a nitrocellulose membrane. Then,hybridization is applied between a fraction and a probe serving as anindicator with RI (radioisotope) to analyze the reaction image of thethus treated fraction by autoradiography.

Measurement, detection and identification of recombinant VZV antigen

Measurement, detection and identification of the viral particles and thevarious antigens produced by the recombinant VZV culture may beconducted by any of the conventional methods usually applied inimmunology and serum diagnosis, such as the RPHA (reversed passivehemagglutination) method using red blood cells coated with an antibody;the ELISA (enzyme-linked immunosorbent assay) or the EIA (enzymeimmunoassay) using an antibody labeled with enzyme, the RIA (radioimmunnoassay) using an antibody labeled with RI, the immunofluorescencemethod consisting of dyeing infected cells with an antibody labeled withFITC (fluorescein isothiocyanate) and determining the presence of anantigen through a fluorescent microscope, and the immuno precipitationmethod comprising fractionating the immuno precipitation reactionproduct between an RI-labeled antigen prepared by culturing arecombinant VZV in a culture medium made by adding and mixing RI-labeledmedium ingredients, on the one hand, and a previously prepared antibody,on the other hand, according to the extent of molecular weight throughSDS-PAGE (SDS-polyacrylamide gel) electrophoresis, then analyzing theelectrophoresis profile by autoradiography. To simplify this process,any of various commercially available measuring kits may be used. Theantigen density can be measured by a conventional method such as thedensity gradient equilibrium method. The shape of antigen particles maybe observed by means of an electron microscope.

Measurement of infectivity of recombinant VZV

Any of the following conventional methods may be adopted; the CPE methodof determining CPE (cell denaturation effect) with the rounding ofinfected cells as the indicator by means of an optical microscops; theplaque assay comprising visually counting PFU (plaque-forming unit) witheach plaque as a unit, which is formed by culturing infected cellsoverlaid by solid culture media containing neutral red and agarose; theplaque assay consisting of visually counting PFU by dyeing infectedcells fixed with formalin with methylene blue solution; and the focusmethod or counting FFU (focus-forming unit) which represents the numberof focuses formed by infected cells by means of an optical microscope.

Cloning of recombinant VZV

Any of the conventional methods may be adopted, such as the focus methodfor cloning of infected cells, and the plaque assay for cloning ofrecombinant VZV. For sampling focuses and plaques, any of glass,plastic, and metal fine cylindrical tubes or cylinders may be used. Thecloned infected cells and recombinant VZVs are inoculated to new cellculture for each clone to culture viruses.

Assay of recombinant VZV immunogenicity

Solution containing a recombinant VZV is inoculated subcutaneously tosmall experimental animals such as monkeys, rabbits, guinea pigs andmice, and then these immunized animals are bred. During the breedingperiod, after inoculation of the virus, the antibody titer of blood ismeasured weekly, monthly or at certain interval, by partially samplingblood in an amount of approximately 3 ml from the vein of the femoralregion in an animal. Measurement of the antibody titer may beaccomplished by any of such conventional methods commonly used forimmunology and serum diagnosis such as the PHA (passivehemagglutination) method using red blood cells coated with the antigenused for immunity, and the neutralization test method between knownviruses in a given amount and the antiserum thereof, in which titer ismeasured by the CPE method or plaque assay, the maximum dilution ofantiserum which neutralizes and reduces the amount of virus by 50%.

Manufacture of live vaccine containing recombinant VZV as effectiveingredient

After culturing a recombinant VZV with the use of human diploid cellculture, the resultant culture is purified by, for example,centrifugation and the recombinant VZV fraction is collected. Solutionsof amino acid and saccharides are added to, and mixed with, the thuscollected fraction, and the resulting solution is diluted to adjust theviral content per dose of live vaccine to at least 1,000 PFU, thusmanufacturing the live vaccine. Samples of the live vaccine aresubjected to various tests regarding safety, effectiveness anduniformity in accordance with the Minimum Requirements for BiologicalProducts (1989) set out in Notification No. 195 of the Japanese Ministryof Health and Welfare, including the "Dried Attenuated Live VaricellaVaccine" and "Recombinant Precipitated Hepatitis B vaccine (from yeast)to confirm and establish eligibility as a vaccine before use. A vaccineis lyophilized in a vial or an ampoule having a volume of approximately3 to 30 ml. It is supplied in an airtight state and stored at atemperature lower than 5° C. The vaccine should be used in compliancewith the instructions given in the Manual: reconstituting thelyophilized content completely with sterilized distilled water beforeuse, and inoculating 0.5 ml per 1 dose subcutaneously.

Manufacture of diagnostic agent containing recombinant VZV antigen aseffective ingredient

After culturing a recombinant VZV with the use of human diploid cellculture, the resultant cell is purified by, for example, centrifugationand the recombinant VZV antigen fraction is collected. Afterinactivating this antigen through, for example, addition of formalin orheating at 56° C., it is diluted and adjusted with, for examples PBS soas to give an antigen protein content of 1 to 10 μg/ml to prepare adiagnostic agent. This antigen is supplied by sealing in a vial or anampoule having a volume of 2 to 50 ml in liquid or dried state. Theantigen is applicable as the antigen for various techniques of serumdiagnosis such as ELISA and PHA, and for the manufacture of antibodies.In the case of a dried product, th e dried content is completelyreconstituted with distilled water before use.

EFFECTS OF THE INVENTION!

(1) Supply of multivalent live vaccine: The recombinant VZV strain madeavailable by the present invention is an attenuated virus withsimultaneously expressing a VZV gene and at least one kind of HBV gene,and furthermore, each expression product has an immunogenicity equal tothat of the currently used vaccine antigen. The expression abilitythereof is genetically stable and satisfactory in the succession ofgenerations of the virus, and this recombinant VZV has safety andeffectiveness as a live vaccine. It is therefore applicable in practiceas an effective ingredient of a multivalent live vaccine or amulti-function live vaccine in place of the conventional mixed orinactivated ones.

(2) Conversion of inactivated vaccine antigen into live vaccine: Somekinds of antigen have been previously used only for an inactivatedvaccine because of the technical restrictions for culturing andpurifying them in addition to the risk of adventitious factors, in spiteof the facts that such vaccine induces only humoral immunity and can notmaintain the immunogenicity for a long period. The present invention, onthe other hand, makes it possible to use such antigens for a livevaccine which can induce both humoral and cellular immunity and has aprolonged immunogenicity on a satisfactory level.

(3) Reduction of manufacturing cost of combined vaccine: A conventionalcombined vaccine has been manufactured by preparing individualingredients of vaccine separately, and then mixing the preparedingredients. According to the present invention, it is possible toprepare two or more kinds of different antigens by culturing recombinantVZVs once, thus permitting minimization of the manufacturing cost ofvaccine.

(4) The present invention permits mass-production of an HBV antigenwhich is otherwise difficult to culture or mas-produce.

(5) It is possible to safely manufacture an antigen against a pathogenhaving a high risk of biohazard.

(6) The recombinant VZV antigen of the present invention, having two ormore different kinds of antigen, is useful also as a multivalentdiagnostic agent capable of simultaneously determining two or more kindsof pathogens. This genomic DNA of the recombinant VZV is applicable forgenetic diagnosis as a chimeric probe consisting of two or moreheterogene DNAS.

(7) Contribution to WHO's EPI (Expansion Program of Immunization): Ashas been described in (1), (2) and (3) above, a multivalent vaccineprovided by this invention must bring considerable advantages to mankindwith its improved and intensified immunity effects, together witheconomic merits and laber savings in the manufacture of vaccines, theadministration for health and hygien, the clinical trials, and forindividuals to be vaccinated. The present invention, therefore, makessignificant contribution to the WHO's proposal for expansion andpromotion of vaccination by using a combined or multivalent vaccine.

Now, the present invention is described in detail by means of examples.It is to be noted that the present invention is not limited to theseexamples.

The phosphate butter saline (PBS) as used in the description of thefollowing examples was prepared as follows: by dissolving 8.0 g NaCl,0.2 g KCl, 2.9 g Na₂ HPO₄. 12H₂ O, 0.2 g KH₂ PO₄, 0.1 g CaCl₂ and 0.1 gMgCl₂. 6H₂ O in distilled water and adjusted to a volume of 1,000 ml.PBS (-) was separately prepared, which did not contain divalent ions,CaCl₂ and MgCl₂.6H₂ O.

EXAMPLE 1 Culture of varicella virus

Human diploid fibroblast, MRC-5, was cultured as the culture host of VZVat 37° C. MEM culture medium manufactured by Gibco Co. (U.S.A.)! wasused as the basic culture medium. Immediately before use, aqueoussolution of 7% (w/v) NaHCO₃ was added to, and mixed with, the culturemedium, and pH was adjusted to 7.4 for the multiplication culture mediumand to 7.8 for the maintenance culture medium. Then, commerciallyavailable fetal bovine serum was further added and mixed so as to give afinal concentration of 10% (v/v) for the muitiplication culture mediumand 3% (v/v) for the maintenance culture medium. The VZV Oka strain(ATCC VR-795) seed virus was inoculated to the thus cultured MRC-5 cellsat 0.01 MOI multiplicity of infection) and cultured for three days at37° C. A maintenance culture medium was used for this culture. DuringVZV culture, the region of infected cells gradually expands along withmultiplication of VZV. A VZV-infected cell, showing rounding under themicroscopic observation, can be detected as an effect known as CPE (celldeterioration effect). By observing the expansion of infected cellsunder the effect of CPE by means of a microscope, therefore, it ispossible to determine the extent of VZV multiplication. VZV culture wascompleted at the moment when CPE was observed over the entire region ofall culture monosheet.

Similarly, Oka strain (WHO Technical Report Series, No. 725, pp.102-124, 1985) seed virus which was a live varicella vaccine strain wascultured. Five bottles each having a culture area of 210 cm² were usedas culture containers. This Oka varicella vaccine strain was employed inall the following examples.

EXAMPLE 2 Extraction and preparation of VZV genomic DNA

Cells infected with the Oka varcella vaccine strain obtained in theExample 1 were collected. More particularly, after the completion ofvirus culture, the culture liquid was thrown away, and PBS (-) solutionof 0.1% (w/v) EDTA-2 Na in an amount of 16 ml was added to the culturebottles and infected cells were detached from the inner wall surface,which were then cooled. Then the pooled cells were centrifuged at 3,000rpm for ten minutes at room temperature, and pellets of infected cellswere collected. The thus collected cells were then added with 3 ml of 10mM Tris-HCl (pH: 8.0) containing 0.5% (v/v) NP40 manufactured by BDHChemicals Co. (UK)! and 10 mM EDTA-2Na to suspend the cells, and thecells were dissolved by holding for 30 minutes at room temperature.Then, to remove cell fragments, the solution was subjected tocentrifugation at 3,000 rpm for 20 minutes at 4° C., and the supernatantwas collected. This cycle of operations was repeated three times, andthe supernatant was pooled. Then, this supernatant was centrifuged on aSW27 rotor (manufactured by Beckman Instruments Co. U.S.A.!) at 27,000rpm for one hour at 4° C., to recover pellets. These pellets weresuspended in 2 ml of TE solution 10 mM Tris-HCl (pH: 8.0), 1 mMEDTA-2Na!. Then, 2 ml of solubilizing liquid 10 mM Tris-HCl (pH: 8.0),1% (w/v) SDS, 10 mM EDTA-2Na, 200 μg/ml Proteinase K, and 100 μg/mlRNase A! was added to the suspension to cause reactions at 37° C.overnight. After the completion of reactions, DNA was extracted twicewith the use of water-saturated phenolchloroform-isoamylalcohol (1:1:1)mixed solution in equal amounts, and the water layer was collected.Then, cold ethanol in a double amount was added to, and mixed with, thiswater layer, and the resulting solution was held overnight at -20° C. tocause precipitation of DNA. After collection by centrifugation, this DNAsuspended in 2 ml of TE solution as described above, and the product wasprovided as a VZV genomic DNA. The concentration of DNA was determinedin terms of absorbance OD₂₆₀.

EXAMPLE 3 Preparation of preS2 and HBs DNA fragment of HBV

Plasmid pM1B11 (Japanese Patent Provisional Publication No. 22,098/88)in which preS1-preS2-HBs gene of HBV's subtype adr strain had beencloned in the BamHI site of pBR322, was digested by the use ofrestriction enzyme HnaII. Then, 0.5% (w/v) agarose gel electronhoresiswas applied, and after collecting HpaII fragment, both ends of thefragment were bluntended with T4 DNA polymerase.

EXAMPLE 4 Cloning of VZV tk gene

The VZV genomic DNA obtained in the Example 2 was digested withrestriction enzyme SacI, and the resultant fragments were subjected to0.5% (w/v) agarose gel electrophoresis to collect an H fragment(Intervirology, 29, 301-310,1988). This DNA fragment was inserted inEnzymology, of plasmid pUC12 (Methods in Enzymology, vol. 101, pp.20-78, Academic Press U.S.A.!, 1983), then transferred to E. coli JM109(ATCC No. 53323), and a plasmid pUC12-tk was prepared. In addition,after digesting pUC12-tk with restriction enzymes EcoRI and FokI,EcoRI-FokI fragments collected through 1.0% (w/v) agarose gelelectrophoresis were cloned again into pUC12, thus obtaining plasmidpEF6 (see FIG. 1).

EXAMPLE 5 Preparation of chimeric plasmid for constructing recombinantVZV

After inserting the HpaII fragment of HBV genomic DNA prepared in theExample 3 into HincII site of the pEF6 tk gene obtained in the Example4, chimeric plasmid pHH was obtained by transferring it to E. coli JM109strain(see FIG. 1). The base sequence at the linkage portion of theinserted HBV gene was determined by means of a 7-DEAZA sequencing kit(manufactured by Takara Shuzo Co.; Nucleic Acid Research, 14, 1319,1988). The result is shown in FIG. 2. In this chimeric pHH, 1,080 basefragments of the HBV's preS2-HBs gene are inserted and linked betweenthe SacI-FokI 4.8 kb fragments of the VZV gene, and the true initiationcodon ATG (methionine) and TCC (serine) of HBV preS2 are transcribed andtranslated in the linked state under the effect of the true promoter andenhancer of the VZV tk gene. In place of the expression of VZV tk gene,27 amino acids of HBV preS2 and all HBs peptide genes are expressed (seeFIGS. 1 and 2).

EXAMPLE 6 Preparation of recombinant VZV

The VZV genomic DNA (2.5 μg/ml) prepared in the Example 2 and thechimeric plasmid pHH (20 μg/ml) prepared in the Example 5 werecontransfected to MRC-5 cells cultured in a plastic petridish having adia. of 60 mm by the calcium phosphate method (Virology, 52, 465-467,1973) (see FIG. 1). Then, the product of cotransfection was cultured,and infected cells were removed by means of a cylinder from each VZVfocus formed through introduction of VZV genome. These infected cellswere inoculated to MRC-5 cells cultured in a 25 cm³ plastic flask foreach focus for multiplication. At the same time, part of the infectedcells were inoculated to MRC-5 cells cultured on a cover slip for eachfocus and were grown, and the result was observed with a microscope bythe immunofluorescence method using anti-HBs monoclonal antibody(manufactured by BML Co. Japan!). More specifically, cells on each coverslip were washed with PBs, and fixed with a mixed solution of coldmethanol and cold acetone in equal amounts at -20° C. or five minutes.Then, these cells were dyed with the above mentioned monoclonal antibodyof mouse and FITC-labeled anti-mouse IgG antibody. Each of the thus dyedsamples was observed by means of a fluorescent microscope to test forthe presence of HBs antigenicity and the intensity thereof with thedegree of fluorescence as the indicator. As a result, five out of 35clones (C1-C35) in total were selected as candidates for recombinantVZV. The ratio of recombination was 14.5%. With reference to the testresult, cell-free VZVs were prepared from infected cells of therecombinant VZV candidata clones from among the infected cellsmultiplicated within the flask as described above. The preparedrecombinant VZVs were stored at -70° C. until practical use thereof(Journal of General Virology, 61, 255-269, 1982). Then, with the use ofthe MRC-5 cells cultured in a 60 mm-dia, plastic petridish, a plaqueassay was carried out on a single clone (C17) having shown the highestdegree of fluorescence from among the above-mentioned candidates forrecombinant VZV. A culture medium 199 (manufactured by Difco Co.U.S.A.!) was used for the plaque assay. In a maintenance culture mediumprepared in the same manner as in the Example 1, a solid culture mediumwas prepared by adding and mixing agarose so as to give a finalconcentration of 0.8% (w/v). This culture medium was used by overlayingon the infected cell monosheet. Dyeing of the infected culture cells wasconducted by adding and mixing neutral red to this solid culture mediumso as to give a final concentration of 0.01% (w/v), and furtheroverlaying the thus prepared culture medium. Virus was inoculated afteradjusting the VZV concentration so that one to two plaques were formedfor each petri dish. Culture was incubated in a 5% (w/v) CO₂ gas chamberwith the other conditions identical with those in the Example 1. VZV wascloned by hooking up each of the expressed plaques individually by meansof a cylinder. One clone (C17-5) from among the obtained recombinantVZVs was inoculated at a rate of one clone infected cell relative tofive to ten non-infected cells, and this was successively conducted fromcell to cell for ten generations. From each of the infected cellcultures of the tenth generation, a cell-free recombinant VZV wasprepared. A strict selection was applied to these recombinant VZVsthrough an immunofluorescence test, and as a result, there wereseparated 20 cell-free recombinant VZV (FIG. 1) clones that produced HBsantigens. These clones were grouped as rVH Oka strain series, and thetwenty strains composing the series were sequentially named in themanner of rVH1 Oka strain, rVH2 Oka strain and rVH3 Oka strain, and fromrVH4 Oka strain to rVH20 Oka strain. As all these twenty strains wereconfirmed to produce HBs antigens to similar extent, production ofantigens from such recombinant VZVs is considered genetically stable andsatisfactory under viral successive culturing.

In the following Examples, rVH7 Oka strain (rVH17-5 Oka strain, ECACCprovisional accession No. V92041523) was used as a typical example ofrecombinant VZV strains falling under the rVH Oka strain series. Theparental strain of rVH7 Oka strain is Oka varicella live vaccine ofattenuated VZV, as described in the Examples 1 and 2.

EXAMPLE 7 Restriction enzyme analysis of recombinant VZV genomic DNA

This analysis was carried out by the Southern blot method (Journal ofMolecular Biology, 98, 503, 1975). After extracting and preparinggenomic DNAs for the Oka varicella live vaccine strain which was theparental strain and the recombinant VZV strain in the same manner as inthe Example 2, the DNA fragment available by digesting each DNA withrestriction enzyme SacI (SstI) was fractionated by 0.8% (w/v) agarosegel electrophoresis, and the resultant gel was dyed with etidiumbromide. Then, a nitrocellulose membrane was placed on top of this gel,and each DNA fraction was transferred to this membrane. Afterhybridization by an RI-labeled probe, autoradiography was applied. Asprobes, DNAs of α-³² P)dCTP-labeled pMlBll and pHH were employed. As aresult, the original SacI-H fragment was detected in the parentalstrain, and the SacI-H' fragment, in the recombinant VZV strain. Becausethe SacI-H fragment contains a VZV tk gene, and no Sacd site is presentin both HBs and tk gene regions of the recombinant VZV strain, theSacI-H fragment of the parental strain is considered to have shifted tothe SacI-H' fragment in the recombinant strain. While the Sacd-Hfragment of the parental strain reacts only with the pHH probe, theSacI-H' fragment of the recombinant strain is hybridized with pMlBll andpHH, respectively. The HBs gene is therefore determined to have beeninserted into the tk gene of the recombinant VZV genome.

EXAMPLE 8 Detection and identification of HBs antigen produced in cellsinfected with recombinant VZV strain

The rVH7 Oka strain obtained in Example 6 was cultured in the samemanner as in Example 1 with the use of five plastic flasks each having aculture area of 150 cm². Resultant supernatants of culture were pooledand centrifuged at 3,000 rpm for 20 minutes at 4° C. Infected cells weredetached from the inner wall surface of the culture flasks with rubberscrubber and suspended in PBS.

(1) Measurement of quantity of produced HBs antigen: Furthercentrifugation was applied at 27,000 rpm for three hours at 4° C. to 35ml of the above-mentioned supernatant, and pellets were collected, whichwere then suspended in 0.2 ml of PBS that formed the culture supernatantfraction of the recombinant strain. In contrast, approximately 10⁶infected cells as described above were suspended in 1 ml of PBS. Afterdestroying the cells by ultrasonic wave (20 kHz, 150 mA, 4 seconds), thesuspension was centrifuged at 3,000 rpm for 20 minutes at 4° C., and theresulting supernatant was collected as the recombinant strain infectedfraction. For the both fractions, the HBs antigen titer was measured bythe application of the RPHA (reverse massive hemagglutination) testusing cells coated with anti-HBs serum. For this test, a commerciallyavailable test kit (manufactured by Midori Juji Co. JAPAN!) was used.For purpose of comparison, there were used also a non-infected cellfraction and a parental strain infected cell fraction prepared fromapproximately 10⁶ cells in the same manner as above, and an HBs antigenproduced in yeast (Japanese Patent Provisional Publication No.22,098/88). These samples were diluted in two stages and subjected tomeasurement. The results are shown in Table 1. Table 1 permitsestimation that the quantity of produced HBs is 10 μg/ml for theinfected cell fraction resulting from culture of the recombinant VZVstrain and 4 μg/ml for the culture supernatant fraction (23 μg/ml forthe original culture supernatant before concentration.)

                  TABLE 1                                                         ______________________________________                                        Measurement of HBs antigen expressed in the recombinant                       VZV-infected culture by the RPHA test                                         Antigen                RPHA value (*)                                         ______________________________________                                        Non-infected cell fraction (10.sup.6 cells/ml)                                                       1.sup.                                                 Parental VZV-infected cell fraction                                                                  1.sup.                                                 (10.sup.6 cells/ml)                                                           Recombinant VZV-infected culture supernatant                                                         2.sup.5                                                (concentrated 175 times)                                                      Recombinant VZV-infected cell fraction                                                               2.sup.6                                                (10.sup.6 cells/ml)                                                           HBs antigen made in yeast (250 μg/ml)                                                             .sup. 2.sup.12                                         ______________________________________                                         (Note) *: Maximum dilution multiples of antigen showing RPHA positivity. 

(2) Detection of HBs antigen by immunofluorescence method: Presence ofany produced HBs antigen was detected in the abovementioned recombinantVZV-infected cells by the application of the immunofluorescence methodusing the monoclonal antibody of HBs antigen as described in the Example6. The result permitted confirmation of the production of HBs antigenwithin the infected cytoplasm.

(3) Confirmation of HBs antigen in culture supernatant by electronmicroscope: The above-mentioned culture supernatant was centrifuged at5,000 rpm for 20 minutes, and then PEG polyethylene glycol) 6,000 wasadded to, and mixed with, this supernatant so as to give a finalconcentration of 10% (w/v) to form a precipitate. Then, precipitatepellets collected through low-speed centrifugation were suspended in TENliquid 20 mM Tris-HCl (pH; 7.4), 1 mM EDTA-2Na, 150 mM NaCl!, and then,this suspension was subjected to CsCl density gradient equilibriumcentrifugation twice by means of an SW27 rotor. With the use of adiscontinued gradient comprising densities of 1.15, 1.25 and 1.4 g/cm³in the centrifugal tube, the precipitate suspension was overlaid on thisCsCl solution, and centrifugation was applied at 23,000 rpm for 20 hoursat 4° C. After the completion of centrifugation, the CsCl solution wasfractionated, and the HBs antigen fraction was determined through theabove-mentioned RPHA test. The resultant HBs antigen fraction was mixedwith TEN liquid, and after adjusting density to 1.2 g/cm³ by CsCl, wasfurther centrifuged by an SW41 at 33,000 rpm for 40 hours at 4° C. Afterthe completion of the centrifugation, the CsCl solution wasfractionated. The HBs antigen fraction was sampled and diluted, andafter overlaying it on a 10% (w/v) sucrose cushion, was centrifuged byan SW41 rotor at 40,000 rpm for three hours at 4° C. The resultantpellets were suspended in PBS and observed through an electronmicroscope. As a result, many particles having a dia. of 20 to 30 nmwere detected in the final purified HBs fraction. It is judged from thisresult that particle-shaped HBs antigen is secreted from infected cells.

(4) Measurement of density of HBs antigen in culture supernatant; Aportion of the purified HBs fraction used in the above-mentionedelectron microscopic observation was fractionated by CsCl densitygradient equilibrium centrifugation of a density of 1.2 g/cm³ at 33,000rpm for 40 hours at 4° C. with the use of an SW41 rotor. Density wascalculated for fractions by measuring the volume and the weight, and atthe same time, the HBs antigen titer was measured by an RPHA test. Theresults showed a density of the purified HBs fraction of 1.20 g/cm³,which represents the peak of the HBs activity (see FIG. 3).

EXAMPLE 9 Identification of VZV and HBs antigens of the recombinant VZVstrain

MRC-5 cells were cultured on three petridishes having a dia. of 100 mmin the same manner as in the Example 1. On one of the three dishes, rVH7Oka strain was inoculated, and the parental strain thereof wasinoculated to the cells on one of the remaining dishes. For cell cultureon the last dish, no virus was inoculated, but the dish was used forpreparing antigen for comparison. These cells were cultured for fourhours in the same manner as in the Example 1, by means of a basicculture medium prepared by adding ³⁵ S! methionine of 50 μCi/ml andcysteine to an MEM culture medium not containing methionine. Then, thecells were detached, and after dissolving the cells into an RIPA buffersolution 20 mM Tris-HCl (pH; 8.0), 1% (w/v) Triton x-100, 0.1% (w/v)SDB, 150 mM NaCl, and 1 mM phenylmethyl-sulfonyl fluoride!, centrifugedat 35,000 rpm for an hour. The supernatant thereof was sampled. Thesupernatant was centrifuged at 3,000 rpm for 20 minutes to collect thesupernatant thereof. Each of the six kinds of supernatant was mixed withanti-VZV guinea-pig serum (Virology, 156, 423-426, 1987), to cause theimmunoprecipitation reaction. Immune complex formed by these reactionsystems were separated individually by column chromatography withprotein A Cepharose CL-4B manufactured by Pharmacia LKB (Sweden)!.Immune complex was similarly formed also between an anti-HBs rabbitserum prepared with the use of an HBs antigen (Japanese PatentProvisional Publication No. 22,098/88) and these six kinds ofsupernatant, and separated. Then, each of these immune complex wassubjected to autoradiography after SDS-PAGE electrophoresis. As aresult, VZV polypeptide was detected simultaneously in the infectedcells of both the parental strain and the recombinant strain. The 26 kand 30 k HBs antigens were detected only in the infected cells of therecombinant strain. In the culture supernatant of the recombinantstrain, the HBs antigens secreted by the infected cells of therecombinant strain described in the Example 8 were identified as boththe 30 k and 35 k polypeptides. It is judged from this identificationthat the 26 k and 30 k HBs antigens synthesized within the infectedcells undergo modification and processing, while being secreted into theculture supernatant, and become the 30 k and 35 k HBs antigens.

EXAMPLE 10 Manufacture of live vaccine containing recombinant VZV aseffective ingredient

After inoculating the rVH7 Oka strain seed virus obtained in the Example6 to MRC-5 cell cultured in twenty bottles each having a culture area of210 cm², culture was conducted in the same manner as in the Example 1.After the completion or the culture, the culture medium was removed, andthe infected cells in the bottles were washed twice with 200 ml ofPBS(-). Then, 20 ml or 0.03% (w/v) EDTA-3Na were added to the infectedcells in the bottles, the cells being detached from the inner wails orthe bottles and suspended. The infected cell suspension in the bottleswas pooled, centrifuged at 2,000 rpm for ten minutes at 4° C., andpellets of the infected cells were collected. The pellets were suspendedagain in 100 ml of PBS(-), and freeze-thawings was done once. Then,after conducting an ultrasonic treatment (20 kHz, 150 mA, 0.3seconds/ml) in an ice-water bath, a centrifugation was applied at 3,000rpm for 20 minutes at 4° C. The supernatant containing cell-free viruswas prepared as the original virus suspension of live vaccine. From thisoriginal virus suspension, 30 ml were sampled for test, and saccharoseand gelatin hydrolysis products dissolved in PBS(-) were added to, andmixed with, the remaining 70 ml of original virus suspension as thevaccine stabilizer so as to give final concentrations of 5% (w/v) and2.5% (w/v) to prepare a final bulk of live vaccine in an amount of 140ml. After sampling 30 ml for test from this final bulk, the remainingbulk was filled into vials each having a capacity of 3 ml by 0.5 ml.After freeze-drying, the bottles were filled with nitrogen gas andplugged with rubber stoppers to seal airtightly the bottles. Thesesubdivided bottles of live vaccine are stored at 4° C., and distilledwater for injection in an amount of 0.5 ml is added to completelyreconstitute the dried content immediately before use. In contrast, 20bottles of the sampled original virus suspension, final bulk andsubdivided product were subjected to various tests. The tests werecarried out in conformity to the Minimum Requirements for BiologicalProducts (1989) "Dried Attenuated Live Varicella Vaccine" prescribed inthe Japanese Ministry of Health and Welfare Notification No. 195, andwith reference to another standard "Recombinant Precipitated Hepatitis BVaccine (originating from yeast)" set forth in the same Notification, toconfirm safety, effectiveness and uniformity and to establisheligibility as a live vaccine. As a result of these tests, theabove-mentioned subdivided product had a virus content of 2×10⁴ PFU/0.5ml and was qualified in the various tests specified in the standard. Ithas therefore been practically used since then as a suitable livevaccine.

EXAMPLE 11 Manufacture of recombinant VZV antigen as diagnostic agent

After inoculating the rVH7 Oka strain seed virus obtained in the Example6 to MRC-5 cell cultures contained in twenty bottles each having aculture area of 210 cm², culture was conducted in the same manner as inthe Example 1. After the completion of culture for a day, culture liquidwas discarded, and the infected cells in the bottles were washed twicewith 200 ml of PBS(-). Then, Phenol Red was removes from Culture Medium199 manufactured by Difco Co. (U.S.A.)! and it was poured into thesebottles, and culture was continued at 37° C. for another Three days.After the completion of the culture, culture liquid was sampled frombottles and pooled. The pooled liquid was then centrifuged at 3,000 rpmfor 20 minutes, and the supernatant was concentrated in volume to 1/20by means of a Minitan Ultra-Filter MW10000 manufactured by Millpore(U.S.A.)!. Then, the concentrated liquid was heated at 56° C. for 30minutes to inactivate the virus. The liquid was diluted with PBS so thatthe content of the recombinant VZV antigen protein in the thus obtainedliquid became 5 μg/ml, and the resultant liquid was poured into ampouleseach having a capacity of 3 ml by 2 ml. The ampoules were sealed bymelting and used as a diagnostic agent of chickenpox and hepatitis B.

EXAMPLE 12 Assay of immunogenicity of recombinant live vaccinecontaining recombinant VZV as an effective ingredient

Immunogenicity of the recombinant varicella live vaccine strainmanufactured in the Example 10 was measured in guinea pigs. Forcomparison, the following five kinds of vaccine were employed: thecommercially available dried attenuated live varicella vaccine which wasthe parental vaccine (prepared by The Research Foundation for MicrobialDiseases of Osaka University), a recombinant precipitated hepatitis Bvaccine one manufactured under Japanese Patent Provisional PublicationNo. 22,098/88, and the other, manufactured by Merck Co. (U.S.A.)! andantigens for comparison prepared through a similar process to that inthe Example 10 from MRC-5 non-infected cells. Each of these vaccines wassubcutaneously inoculated to three guinea pigs of a three weeks old,average body weight of 250 g. The recombinant strain and parental strainvaccines were diluted with PBS(-) so that the amount of inoculationbecame 3,000 PFU or 2,000 PFU/dose. The hepatitis B vaccines wereinoculated so as to give an amount of HBs antigen of 10 μg/animal. Four,six and eight weeks after the vaccine inoculation, blood was partiallycollected from the vein of the femoral region of each of the inoculatedanimals to measure the antibody titer of blood. For the measurement ofthe antibody titer, the neutralization test method (Journal of GeneralVirology, 61, 255-269, 1982) was adopted for VZV, and the PHA (passivehemaggloutination) test kit (manufactured by the Chemical and SerumTherapy Research Laboratory) using cells coated with HBs antigen, forHBs. The results are shown in Tables 2 and 3. The parental strain andthe recombinant strain vaccines induced the VZV antibody on the samelevel, whereas an HBs antibody was detected only in the recombinantstrain vaccine inoculation (see Table 2). Furthermore, theimmunogenicity of the recombinant strain vaccine against HBs was equalto that of the commercially available hepatitis B vaccine (see Table 3).It is judged from these results that the recombinant strain vaccine hasexcellent immunogenicity against both the VZV and Has antigens.

                  TABLE 2                                                         ______________________________________                                        Antibody response of guinea-pigs immunized by VZV parental                    strain and recombinant strain vaccines against VZV and HBs antigens                           Anti-VZV  Anti-HBs antigen                                    Immunogen       antibody titer                                                                          antibody titer                                      ______________________________________                                        VZV-HBs recombinant                                                           strain vaccine                                                                Subject No. 1   8         8                                                   No. 2           4         4                                                   No. 3           8         4                                                   VZV parental                                                                  strain vaccine                                                                Subject No. 4   4         <1                                                  No. 5           8         <1                                                  No. 6           8         <1                                                  Non-infected cell                                                             antigen for comparison                                                        Subject No. 7   <1        <1                                                  No. 8           <1        <1                                                  No. 9           <1        <1                                                  ______________________________________                                         (Note 1) Amount of subcutaneous inoculation of vaccine: 2,000 PFU of          cellfree virus/animal.                                                        (Note 2) Measurement of antibody titer: conducted by collecting blood fou     weeks after vaccine inoculation.                                              (Note 3) Method of measurement of antibody titer: the neutralization test     was adopted for VZV, and PHA, for HBs.                                        (Note 4) Figures: Maximum dilution of immunized rabbit serum showing          positive antibody reaction.                                              

                  TABLE 3                                                         ______________________________________                                        Comparison of immunity response of guinea pigs                                inoculated with recombinant VZV strain vaccine and                            existing recombinant HBs vaccine against HBs                                             Test 1*         Test 2*                                                         4       6         8     4                                        Immunogen    weeks   weeks     weeks weeks                                    ______________________________________                                        VZ-HBs                                                                        recombinant vaccine                                                           Subject No. 10                                                                             32      64        64    32                                       No. 11       32      64        64    32                                       No. 12       64      64        64    128                                      Alum precipitation                                                            hepatitis B vaccine                                                           Subject No. 13                                                                             32      32        32    128                                      No. 14       128     128       128   32                                       No. 15       32      32        32    64                                       VZV parental                                                                  strain vaccine                                                                Subject No. 16                                                                             <1      <1        <1    NT                                       No. 17       <1      <1        <1    NT                                       No. 18       <1      <1        <1    NT                                       ______________________________________                                         (Note 1) Amount or subcutaneous inoculation of vaccione:                      *In test 1, 10 μg HBs antigen/animal, using a recombinant HBs vaccine      (Japanese Patent Provisional Publication No. 22,098/88);                      *In test 2, 10 μg HBs antigen/animal, using a recombinant HBs vaccine      (manufactured by Merck);                                                      *In both tests 1 and 2, 3,000 PFU cellfree virus/animal, using both the       VZV recombinant and VZV parental strain vaccines;                             (Note 2) Measurement of antibody titer: After immunizing, blood was           sampled following the lapse of weeks as listed in the table, and              measurement was based on PHA.                                                 (Note 3) Figures: Maximum dilution of immunized rabbit serum showing          positive antibody reaction;                                                   (Note 4) NT: no test was effected.                                       

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (iii) HYPOTHETICAL:                                                           (iv) ANTI-SENSE:                                             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CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (iii) HYPOTHETICAL:                                                           (iv) ANTI-SENSE:                                                              (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (B) STRAIN:                                                                   (C) INDIVIDUAL ISOLATE:                                                       (D) DEVELOPMENTAL STAGE:                                                      (E) HAPLOTYPE:                                                                (F) TISSUE TYPE:                                                              (G) CELL TYPE:                                                                (H) CELL LINE:                                                                (I) ORGANELLE:                                                                (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY:                                                                  (B) CLONE:                                                                    (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT:                                                       (B) MAP POSITION:                                                             (C) UNITS:                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:                                                                  (B) TITLE:                                                                    (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO:                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AAACATGTCCGGA13                                                               __________________________________________________________________________

What is claimed is:
 1. An antigenic hepatitis B virus protein, which isobtained from a cell infected with a recombinant varicella-zoster virusor is obtained from a culture supernatant of said infected cell, whereinthe recombinant varicella-zoster virus comprises the genomic DNA of theOka varicella vaccine strain and the HpaII fragment of the hepatitis Bsurface antigen gene, wherein said fragment is inserted in the HincIIsite of the thymidine kinase gene of said genomic DNA, so that the5'-end of said fragment is linked with the starting codon of saidthymidine kinase gene in a correct reading frame, and the hepatitis Bsurface antigen is expressed under sole control of said thymidine kinasegene promoter.
 2. The antigenic protein according to claim 1, which hasa molecular weight of about 26, 30 or 35 kilo-daltons.
 3. The antigenicprotein according to claim 1, wherein the recombinant varicella-zostervirus is rVH17-5 Oka strain (ECACC No. V92041523).
 4. A diagnostic agentwhich detects antibody against hepatitis B virus, said agent containingthe antigenic protein as claimed in claim
 1. 5. A diagnostic agent whichdetects antibody against hepatitis B virus, said agent containing theantigenic protein as claimed in claim
 2. 6. A diagnostic agent whichdetects antibody against hepatitis B virus, said agent containing theantigenic protein as claimed in claim 3.